John Anderson

Human γδ T Lymphocytes Are Licensed for Professional Antigen Presentation by Interaction with Opsonized Target Cells

Activated human blood γδ T cells have also been previously demonstrated to behave as professional APCs, although the processes that control APC function have not been characterized. n this study, we show that the acquisition of potent APC function by human blood γδ T cells is achieved after physical interaction with an Ab-coated target cell, a process that we refer to as licensing. In cancer models, licensing of γδ T cells by tumor-reactive mAbs promotes the uptake of tumor Ags and professional presentation to tumor-reactive αβ T cells. We propose that licensing by Ab is a mechanism whereby the adaptive properties of γδ T cells are induced by their innate functions in a spatially and temporally controlled manner.

Neuroblastoma arginase activity creates an immunosuppressive microenvironment that impairs autologous and engineered immunity

Neuroblastoma is the most common extracranial solid tumor of childhood, and survival remains poor for patients with advanced disease. Novel immune therapies are currently in development, but clinical outcomes have not matched preclinical results. Here, we describe key mechanisms in which neuroblastoma inhibits the immune response. We show that murine and human neuroblastoma tumor cells suppress T-cell proliferation through increased arginase activity. Arginase II is the predominant isoform expressed and creates an arginine-deplete local and systemic microenvironment. Neuroblastoma arginase activity results in inhibition of myeloid cell activation and suppression of bone marrow CD34(+) progenitor proliferation. Finally, we demonstrate that the arginase activity of neuroblastoma impairs NY-ESO-1-specific T-cell receptor and GD2-specific chimeric antigen receptor-engineered T-cell proliferation and cytotoxicity. High arginase II expression correlates with poor survival for patients with neuroblastoma. The results support the hypothesis that neuroblastoma creates an arginase-dependent immunosuppressive microenvironment in both the tumor and blood that leads to impaired immunosurveillance and suboptimal efficacy of immunotherapeutic approaches.

Lack of T-cell responses following autologous tumour lysate pulsed dendritic cell vaccination, in patients with relapsed osteosarcoma.

BackgroundImmunotherapy using autologous dendritic cell (DC) vaccination has not been systematically evaluated in osteosarcoma. We therefore conducted a phase I trial to assess feasibility, safety and tumour-specific immune responses in patients with relapsed disease.Patients and methodsOf 13 recruited patients with relapsed osteosarcoma, 12 received 3 weekly vaccines of autologous DCs matured with autologous tumour lysate and keyhole limpet haemocyanin (KLH), to a maximum of 6 vaccinations. An additional 3 paediatric patients afflicted with other tumour types and with relapsed disease received vaccines generated with identical methodology. Immune responses were assessed using an ELISpot assay for the detection of interferon gamma, whilst interleukin-2 and granzyme B were additionally assessed in cases where interferon-γ responses were induced.ResultsIn total 61 vaccines, of homogeneous maturation phenotype and viability, were administered with no significant toxicity. Only in 2 out of 12 treated osteosarcoma cases was there an induction of specific T-cell immune response to the tumour, whilst a strong but non-specific immune response was induced in 1 further osteosarcoma patient. Immune response against KLH was induced in only 3 out of 12 osteosarcoma patients. In contrast, three additional non-osteosarcoma patients showed significant T-cell responses to vaccine.ConclusionWe have shown the strategy of DC vaccination in relapsed osteosarcoma is safe and feasible. However, significant anti-tumour responses were induced in only 2 out of 12 vaccinated patients with no evidence of clinical benefit. Comparison of results with identically treated control patients suggests that osteosarcoma patients might be relatively insensitive to DC-based vaccine treatments.

Increased PRAME antigen-specific killing of malignant cell lines by low avidity CTL clones, following treatment with 5-Aza-2′-Deoxycytidine

The cancer testis antigen Preferentially Expressed Antigen of Melanoma (PRAME) is overexpressed in many solid tumours and haematological malignancies whilst showing minimal expression in normal tissues and is therefore a promising target for immunotherapy. HLA-A0201-restricted peptide epitopes from PRAME have previously been identified as potential immunogens to drive antigen-specific autologous CTL responses, capable of lysing PRAME expressing tumour cells. CTL lines, from 13 normal donors and 10 melanoma patients, all of whom were HLA-A0201 positive, were generated against the PRAME peptide epitope PRA100−108. Specific killing activity against PRA100−108 peptide-pulsed targets was weak compared with CTL lines directed against known immunodominant peptides. Moreover, limiting dilution cloning from selected PRAME-specific CTL lines resulted in the generation of a clone of only low to intermediate avidity. Addition of the demethylating agent 5-aza-2′-Deoxycytidine (DAC) increased PRAME expression in 7 out of 11 malignant cell lines including several B lineage leukaemia lines and also increased class I expression. Pre-treatment of target cells was associated with increased sensitivity to antigen-specific killing by the low avidity CTL. When CTL, as well as of the target cells, were treated, the antigen-specific killing was further augmented. Interestingly, one HLA-A0201-negative DAC-treated line (RAJI) showed increased sensitivity to killing by clones despite a failure of expression of PRAME or HLA-A0201. Together these data point to a general increased augmentation of cancer immunogenocity by DAC involving both antigen-specific and non-specific mechanisms.

Pilot study of F18-Fluorodeoxyglucose Positron Emission Tomography/computerised tomography in Wilms’ tumour: Correlation with conventional imaging, pathology and immunohistochemistry

Wilms tumour is the second most common paediatric solid tumour. Prognosis is good although higher stage disease carries significant mortality and treatment related morbidity. In the UK, risk stratification is based on histological response to pre-operative chemotherapy. F(18)-Fluorodeoxyglucose Positron Emission Tomography (F(18)FDG-PET) is an emerging functional imaging technique in paediatric oncology. Little is known about the relationship between F(18)FDG-PET images and the disease process of Wilms tumour. We performed F(18)FDG-PET/CT scans in seven children with Wilms tumour after induction chemotherapy, immediately before surgery. The standard uptake values (SUV) of F(18)FDG-PET/CT images were related to conventional imaging and histopathological findings. In total seven children were studied. F(18)FDG-PET/CT was consistently safely performed. All tumours showed F(18)FDG activity. Four tumours had activity with SUV/bw max >5 g/ml. Histological examination of these active areas revealed viable anaplastic Wilms tumour. Furthermore, in these four tumours GLUT-1 and Ki67 immunostaining was strongly positive. Three further tumours demonstrated lower uptake (SUV/bw max <5 g/ml), which represented areas of microscopic foci of residual viable tumour mixed with post chemotherapy change. Metastatic disease was F(18)FDG avid in two of four children with stage four diseases. In conclusion, following chemotherapy, active Wilms tumour is F(18)FDG avid and higher SUV was seen in histologically high risk disease.

Ultrasound-guided core needle biopsy for the diagnosis of rhabdomyosarcoma in childhood

Most commonly a tissue diagnosis of rhabdomyosarcoma (RMS) in children is made by biopsy as opposed to primary resection. Open surgical procedures are often recommended to obtain sufficient material for accurate and complete diagnostic work up. Our institution has routinely used image‐guided needle biopsies for soft tissue tumour diagnosis. We therefore sought to assess diagnostic accuracy and completeness, and procedure safety of consecutive patients diagnosed by needle biopsies in a single institution.

An Optimized GD2-Targeting Retroviral Cassette for More Potent and Safer Cellular Therapy of Neuroblastoma and Other Cancers

Neuroblastoma is the commonest extra cranial solid cancer of childhood. Despite escalation of treatment regimens, a significant minority of patients die of their disease. Disialoganglioside (GD2) is consistently expressed at high-levels in neuroblastoma tumors, which have been targeted with some success using therapeutic monoclonal antibodies. GD2 is also expressed in a range of other cancer but with the exception of some peripheral nerves is largely absent from non-transformed tissues. Chimeric Antigen Receptors (CARs) are artificial type I proteins which graft the specificity of a monoclonal antibody onto a T-cell. Clinical data with early CAR designs directed against GD2 have shown some promise in Neuroblastoma. Here, we describe a GD2-targeting CAR retroviral cassette, which has been optimized for CAR T-cell persistence, efficacy and safety.

Avoidance of On-Target Off-Tumor Activation Using a Co-stimulation-Only Chimeric Antigen Receptor

Chimeric antigen receptors (CARs) combine Txa0cell activation with antibody-mediated tumor antigen specificity, bypassing the need for Txa0cell receptor (TCR) ligation. A limitation of CAR technology is on-target off-tumor toxicity caused by target antigen expression on normal cells. Using GD2 as a model cancer antigen, we hypothesized that this could be minimized by using Txa0cells expressing Vγ9Vδ2 TCR, which recognizes transformed cells in a major histocompatibility complex (MHC)-unrestricted manner, in combination with a co-stimulatory CAR that would function independently of the TCR. An anti-GD2 CAR containing a solitary endodomain derived from the NKG2D adaptor DAP10 was expressed in Vγ9Vδ2+ Txa0cells. Differential ligation of the CAR and/or TCR using antibody-coated beads showed that pro-inflammatory cytokine response depended on activation of both receptors. Moreover, in killing assays, GD2-expressing neuroblastoma cells that engaged the Vγ9Vδ2 TCR were efficiently lysed, whereas cells that expressed GD2 equivalently but did not engage the Vγ9Vδ2 TCR were untouched. Differentiation between X-on tumor and X-off tumor offers potential for safer immunotherapy and broader target selection.

AN AUTOMATED TECHNIQUE FOR THE ACCURATE POSITIONING OF SEDIMENT CORE SITES AND THE BATHYMETRIC MAPPING OF LAKE BASINS

Automated techniques for sediment core site positioning, hydrographic survey, and contour mapping are described. The computer contouring system can be extended to calculate values for morphometric variables such as maximum length (1max), maximum width (wmax), lake perimeter (10), lake area (A) and lake volume (V). The techniques are exemplified using results from Augher Lough, Northern Ireland.

Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewings sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewings sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewings sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewings sarcoma both rational and feasible.